PCR assay of the molecular mutation at cinnabar and vestigial genes - - PowerPoint PPT Presentation

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PCR assay of the molecular mutation at cinnabar and vestigial genes - - PowerPoint PPT Presentation

Nov 06, 2023 146 likes •381 views

PCR assay of the molecular mutation at cinnabar and vestigial genes of Drosophila melanogaster germ cells after -rays action Aleksievich Olga, BSU Igor D. Alexandrov, Ph. D., Furs Olga, BSU Dr. Sci. (Biology), chief.

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SLIDE 1

PCR assay of the molecular mutation at cinnabar and vestigial genes

  • f Drosophila melanogaster

germ cells after γ-rays action

  • Aleksievich Olga, BSU
  • Furs Olga, BSU
  • Sivak Tatiana, BSU
  • Sharash Irina, BSU
  • Igor D. Alexandrov, Ph. D.,
  • Dr. Sci. (Biology), chief.

Sci.res.

  • Margarita V. Alexandrova,
  • Ph. D., senior sci. res.
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SLIDE 2

Project aim Project aim

TO STUDY THE MOLECULAR GENETIC ACTION OF GAMMA-RAYS ON THE CINNABAR AND VESTIGIAL GENES IN DROSOPHILA MELANOGASTER GERM CELLS

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Drosophila melanogaster, species is commonly known as the common fruit fly or vinegar fly. One of the best model organism in genetics and radiobiology because:

§ Well studied example,

gene structure known

§

Has common principal DNA structure with humans

§

Short life cycle (~10 days)

§

Permits the study of heritable gene mutation

§ Low cost

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SLIDE 4

Wild phenotype of Drosophila

melanogaster

Phenotype of cinnabar gene mutants Phenotype of vestigial gene mutants γ-rays γ-rays

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SLIDE 5

Main steps Main steps

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SLIDE 6

DNA isolation DNA isolation

  • cell lysis
  • DNA sorbtion with Silica Solution
  • DNA purification using washing buffer
  • DNA extraction using Extra Gene
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SLIDE 7

Polymerase Chain Reaction - PCR

  • to amplify specific

fragment of DNA

  • method based on

in vitro replication of DNA using:

Taq polymerase

primers dNTP reaction buffer

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SLIDE 8

Electrophoresis

! !

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SLIDE 9

Visualization

  • using ethidium bromide
  • dye intercalates beetween

bases UV UV

UV UV

red red-

  • orange
  • range

AB Max EM Max

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SLIDE 10

Schem e of ves t i gi al gene

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SLIDE 11

Ex 6-8 Ex 5 Ex 3 Ex 4 Ex 1 Ex 2

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SLIDE 12

in 2-2 in 1 in 3

in 2-3 in 2-4

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V es t i gi al m ut ant s w i t h génot ype Phr

№

Mutation code Dose ex1 (983b) ex2 (777b) ex3 (471b) ex4 (381b) ex5 (670b) ex6-8 (768b) 1

Phr14

+ + + + + + 2

Phr16

+ + + + + + 3

vg89e19

γ, 40

+ + + + + + 4

vg89e20

γ, 40

+ + + + + + 5

vg89e23

γ, 40

+ + + + + + 6

vg89e47

γ, 40 + + + + + + 7

vg89e64a

γ, 40

+ + + + + + 8

vg89e88

γ, 40

+ + + + + + 9

vg89e104b

γ, 40

+ + + + + +

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SLIDE 14

Mutation code Dose in1 (983b) In2-2 (471b) In2-3 (381b) In2-4 (670b) in3 (768b)

№

1 8 10 15 16 1

Phr14

+ + + + + 2

Phr16

+ + + + + 3

vg89e19

γ, 40

+ + + + + 4

vg89e20

γ, 40

+ + + + + 5

vg89e23

γ, 40

+ + + + + 6

vg89e47

γ, 40 + + + + insertion 7

vg89e64a

γ, 40

+ + + + + 8

vg89e88

γ, 40

+ + + + + 9

vg89e104b

γ, 40

+ + + + +

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SLIDE 15

Res ul t s

§ 2 control lines and 7 mutants (γ-irradiated) were

examined

§ 8 exones and 3 intrones of vestigial gene were

analyzed

§ 1 insertion was detected in 3d introne of 1 vestigial

gene mutant

§ More than 60 polymerase chain reactions were carried

  • ut
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Concl us i ons

§ Such a little quantity of PCR-detected deletions could

be caused by specific action of γ-rays. Because the type of radiation mentioned above induce point damage (as a rule) that cannot be detected by PCR

§ Mutant phenotype can be caused by changes in exones

and intrones of the gene

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St r uct ur e of t he ci nnabar gene

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Vi s ual i z at i on of El e ct r ophore s i s Us i ng UV Li ght

Fragment 1 Fragment 2 Fragment 3

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PCR r es ul t s of t he 1s t , 2nd and 3t h f r agm ent s of t he ci nnabar gene

№ Dose № of fond № of mutations Genotype Fragments 1st 2hd 3th 1 40 Gy γ Cn3 Cn[74b2]vg/ Cy2004 D-18 + + + 2 40 Gy γ Cn4 Cn[74b3]/Cy0 D-18 + + + 3 40 Gy γ Cn5 Cn[74b4] /Cy204 D-32 + + + 4 40 Gy γ Cn36 Cn[78k1]/Cy204 D-32 + + + 5 40 Gy γ Cn78 Cn[81l4] /Cy2004 D-32 + + + 6 40 Gy γ Cn-n83+In Cn[83b27] /Cy204 D-32 + + + 7 20Gy γ Cn114 Cn[87l80]vg/ Cy2004 D-32 + + + 8 10Gy γ Cn122 Cn[87f149]/ Cy204 D-32 + + + 9 60Gy γ Cn140 Cn[88d7]/CyO D-32 + + + 10 2,5 Gy,12C Cn198 Cn[10d3-4]/Cy204 D-18 +

  • +
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Concl us i ons

Gamma-rays induce point damage (as a rule) that cannot be detected by PCR Only 1 PCR-detected deletion was observed. It can be caused by high-dense irradiation of the mutant

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G ener al Concl us i ons

§ We have studied the molecular alterations induced

by ionizing radiation at 2 Drosophila genes with different sizes, structure and position on the chromosomes.

§ Two different genes-targets react in the same way

  • n the action of radiation. It is suggested that

different position of 2 genes on the chromosome and difference in sizes don’t influence on their radiomutability.

§ Mutations can be caused by changes in intrones and

exones

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Thanks f or your at t ent i on!